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Charges in lipid head groups generate electrical surface potentials at cell membranes, and changes in their composition are involved in various signaling pathways, such as T-cell activation or apoptosis. Here, we present a DNA origami-based sensor for membrane surface charges with a quantitative fluorescence read-out of single molecules. A DNA origami plate is equipped with modifications for specific membrane targeting, surface immobilization, and an anionic sensing unit consisting of single-stranded DNA and the dye ATTO542. This unit is anchored to a lipid membrane by the dye ATTO647N, and conformational changes of the sensing unit in response to surface charges are read out by fluorescence resonance energy transfer between the two dyes. We test the performance of our sensor with single-molecule fluorescence microscopy by exposing it to differently charged large unilamellar vesicles. We achieve a change in energy transfer of ∼10% points between uncharged and highly charged membranes and demonstrate a quantitative relation between the surface charge and the energy transfer. Further, with autocorrelation analyses of confocal data, we unravel the working principle of our sensor that is switching dynamically between a membrane-bound state and an unbound state on the timescale of 1-10 ms. Our study introduces a complementary sensing system for membrane surface charges to previously published genetically encoded sensors. Additionally, the single-molecule read-out enables investigations of lipid membranes on the nanoscale with a high spatial resolution circumventing ensemble averaging.

A rectangular DNA origami structure is one of the most studied and often used motif for applications in DNA nanotechnology. Here, we present two assays to study structural changes in DNA nanostructures and reveal a reversible rolling-up of the rectangular DNA origami structure induced by bivalent cations such as magnesium or calcium. First, we applied one-color and two-color superresolution DNA-PAINT with protruding strands along the long edges of the DNA origami rectangle. At increasing salt concentration, a single line instead of two lines is observed as a first indicator of rolling-up. Two-color measurements also revealed different conformations with parallel and angled edges. Second, we placed a gold nanoparticle and a dye molecule at different positions on the DNA origami structure. Distance dependent fluorescence quenching by the nanoparticle reports on dynamic transitions as well as it provides evidence that the rolling-up occurs preferentially along the diagonal of the DNA origami rectangle. The results will be helpful to test DNA structural models and the assays presented will be useful to study further structural transitions in DNA nanotechnology.

Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches including optical voltage-sensing dyes and genetically encoded voltage indicators have evolved. Here, we present a DNA nanotechnology-based system. Using DNA origami, we incorporate and optimize different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we use a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA connecting the DNA origami and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit is read out by changes of Fluorescence Resonance Energy Transfer (FRET) of single sensors attached to liposomes. They show a FRET change of ∼5% for ΔΨ=100 mV and allow adapting the potential range of highest sensitivity. Further, the working mechanism is rationalized by molecular dynamics simulations. Our approach holds potential for the application as non-genetically encoded sensors at membranes.

Despite the thorough investigation of graphene since 2004, altering its surface chemistry and reproducible functionalization remain challenging. This hinders fabrication of more complex hybrid materials with controlled architectures, and as a consequence the development of sensitive and reliable sensors and biological assays. In this contribution, we introduce DNA origami structures as nanopositioners for placing single dye molecules at controlled distances from graphene. The measurements of fluorescence intensity and lifetime of single emitters carried out for distances ranging from 3 to 58 nm confirmed the d–4 dependence of the excitation energy transfer to graphene. Moreover, we determined the characteristic distance for 50% efficiency of the energy transfer from single dyes to graphene to be 17.7 nm. Using pyrene molecules as a glue to immobilize DNA origami nanostructures of various shape on graphene opens new possibilities to develop graphene-based biophysics and biosensing.

Smartphone-based fluorescence microscopy has been rapidly developing over the last few years, enabling point-of-need detection of cells, bacteria, viruses, and biomarkers. These mobile microscopy devices are cost-effective, field-portable, and easy to use, and benefit from economies of scale. Recent developments in smartphone camera technology have improved their performance, getting closer to that of lab microscopes. Here, we report the use of DNA origami nanobeads with predefined numbers of fluorophores to quantify the sensitivity of a smartphone-based fluorescence microscope in terms of the minimum number of detectable molecules per diffraction-limited spot. With the brightness of a single dye molecule as a reference, we compare the performance of color and monochrome sensors embedded in state- of-the-art smartphones. Our results show that the monochrome sensor of a smartphone can achieve better sensitivity, with a detection limit of ∼10 fluorophores per spot. The use of DNA origami nanobeads to quantify the minimum number of detectable molecules of a sensor is broadly applicable to evaluate the sensitivity of various optical instruments.

Graphene exhibits outstanding fluorescence quenching properties that can become useful for biophysics and biosensing applications, but it remains challenging to harness these advantages due to the complex transfer procedure of chemical vapor deposition-grown graphene to glass coverslips and the low yield of usable samples. Here, we screen 10 graphene-on-glass preparation methods and present an optimized protocol. To obtain the required quality for single-molecule and super-resolution imaging on graphene, we introduce a graphene screening method that avoids consuming the investigated sample. We apply DNA origami nanostructures to place fluorescent probes at a defined distance on top of graphene-on-glass coverslips. Subsequent fluorescence lifetime imaging directly reports on the graphene quality, as deviations from the expected fluorescence lifetime indicate imperfections. We compare the DNA origami probes with conventional techniques for graphene characterization, including light microscopy, atomic force microscopy, and Raman spectroscopy. For the latter, we observe a discrepancy between the graphene quality implied by Raman spectra in comparison to the quality probed by fluorescence lifetime quenching measured at the same position. We attribute this discrepancy to the difference in the effective area that is probed by Raman spectroscopy and fluorescence quenching. Moreover, we demonstrate the applicability of already screened and positively evaluated graphene for studying single-molecule conformational dynamics on a second DNA origami structure. Our results constitute the basis for graphene-based biophysics and super-resolution microscopy.

Summary: DNA nanotechnology offers new biosensing approaches by templating different sensor and transducer components. Here, we combine DNA origami nanoantennas with label-free antibody detection by incorporating a nanoswitch in the plasmonic hotspot of the nanoantenna. The nanoswitch contains two antigens that are displaced by antibody binding, thereby eliciting a fluorescent signal. Single-antibody detection is demonstrated with a DNA origami integrated anti-digoxigenin antibody nanoswitch. In combination with the nanoantenna, the signal generated by the antibody is additionally amplified. This allows the detection of single antibodies on a portable smartphone microscope. Overall, fluorescence-enhanced antibody detection in DNA origami nanoantennas shows that fluorescence-enhanced biosensing can be expanded beyond the scope of the nucleic acids realm.

We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule localization method with a simplified experimental setup and additional fluorescence lifetime information. In contrast to the original MINFLUX implementation, p-MINFLUX uses interleaved laser pulses to deliver the doughnut-shaped excitation foci at a maximum repetition rate. Using both static and dynamic DNA origami model systems, we demonstrate the performance of p-MINFLUX for single-molecule localization nanoscopy and tracking, respectively. p-MINFLUX delivers 1-2 nm localization precision with 2000-1000 photon counts. In addition, p-MINFLUX gives access to the fluorescence lifetime enabling multiplexing and super-resolved lifetime imaging. p-MINFLUX should help to unlock the full potential of innovative single-molecule localization schemes. Fil: Masullo, Luciano Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Steiner, Florian. Ludwig Maximilians Universitat; Alemania Fil: Zähringer, Jonas. Ludwig Maximilians Universitat; Alemania Fil: Lopez, Lucía Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Bohlen, Johann. Ludwig Maximilians Universitat; Alemania Fil: Richter, Lars. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Cole, Fiona. Ludwig Maximilians Universitat; Alemania Fil: Tinnefeld, Philip. Ludwig Maximilians Universitat; Alemania Fil: Stefani, Fernando Daniel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina

DNA nanotechnology and advances in the DNA origami technique have enabled facile design and synthesis of complex and functional nanostructures. Molecular devices are, however, prone to rapid functional and structural degradation due to the high proportion of surface atoms at the nanoscale and due to complex working environments. Besides stabilizing mechanisms, approaches for the self‐repair of functional molecular devices are desirable. Here we exploit the self‐assembly and reconfigurability of DNA origami nanostructures to induce the self‐repair of defects of photoinduced and enzymatic damage. We provide examples of repair in DNA nanostructures showing the difference between unspecific self‐regeneration and damage specific self‐healing mechanisms. Using DNA origami nanorulers studied by atomic force and superresolution DNA PAINT microscopy, quantitative preservation of fluorescence properties is demonstrated with direct potential for improving nanoscale calibration samples., Self‐assembly and reconfigurability of DNA origami is exploited to realize self‐repairing functional nanodevices. A self‐regenerating brightness label is realized by constant exchange of labeling strands from solution. Structural self‐healing of a DNA origami nanoruler is achieved by excess of staple strands that exchange with enzymatically or photo‐induced damaged strands.

In recent years, DNA origami nanorulers for superresolution (SR) fluorescence microscopy have been developed from fundamental proof-of-principle experiments to commercially available test structures. The self-assembled nanostructures allow placing a defined number of fluorescent dye molecules in defined geometries in the nanometer range. Besides the unprecedented control over matter on the nanoscale, robust DNA origami nanorulers are reproducibly obtained in high yields. The distances between their fluorescent marks can be easily analysed yielding intermark distance histograms from many identical structures. Thus, DNA origami nanorulers have become excellent reference and training structures for superresolution microscopy. In this work, we go one step further and develop a calibration process for the measured distances between the fluorescent marks on DNA origami nanorulers. The superresolution technique DNA-PAINT is used to achieve nanometrological traceability of nanoruler distances following the guide to the expression of uncertainty in measurement (GUM). We further show two examples how these nanorulers are used to evaluate the performance of TIRF microscopes that are capable of single-molecule localization microscopy (SMLM).

Graphene is considered a game-changing material, especially for its mechanical and electrical properties. This work exploits that graphene is almost transparent but quenches fluorescence in a range up to ≈40 nm. Graphene as a broadband and unbleachable energy-transfer acceptor without labeling, is used to precisely determine the height of molecules with respect to graphene, to visualize the dynamics of DNA nanostructures, and to determine the orientation of Forster-type resonance energy transfer (FRET) pairs. Using DNA origami nanopositioners, biosensing, single-molecule tracking, and DNA PAINT super-resolution with

Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the bio-molecule(s) of interest to a biocompatible surface. Here we describe a robust surface passivation method by dense polymer-brush grafting, based on optimized polyethylene-glycol (PEG) deposition conditions, exactly at the lower critical point of an aqueous biphasic PEG-salt system. The increased biocompatibility achieved, compared to PEG deposition in sub-optimal conditions away from the critical point, allowed us to successfully detect the assembly and function of a large macro-molecular machine, a fluorescent-labeled multi-subunit, human RNA Polymerase II Transcription Pre-Initiation Complex, on single, promoter-containing, surface-immobilized DNA molecules. This platform will enable probing the complex biochemistry and dynamics of large, multi-subunit macromolecular assemblies, such as during the initiation of human RNA Pol II transcription, at the single-molecule level.

Most measurements of fluorescence lifetimes on the single-molecule level are carried out using avalanche photon diodes (APDs). These single-photon counters are inherently slow, and their response shows a strong dependence on photon energy, which can make reconvolution of the instrument response function (IRF) challenging. An ultrafast time resolution in single-molecule fluorescence is crucial, e.g., in determining donor lifetimes in donor-acceptor couples which undergo energy transfer, or in plasmonic antenna structures, where the radiative rate and non-radiative rates are enhanced. We introduce a femtosecond double-excitation (FeDEx) photon correlation technique, which measures the degree of photon antibunching as a function of time delay between two excitation pulses. In this boxcar integration, the time resolution of fluorescence transients is limited solely by the laser pulse length and is independent of the detector IRF. The versatility of the technique is demonstrated with a custom-made donor-acceptor complex with one donor and two acceptors and with single dye molecules positioned accurately between two gold nanoparticles using DNA origami. The latter structures show ∼75-fold radiative-rate enhancement and fluorescence lifetimes down to 19 ps, which is measured without the need for any reconvolution. With the potential of measuring subpicosecond fluorescence lifetimes, plasmonic antenna structures can now be optimized further.

An ideal point light source is as small and as bright as possible. For fluorescent point light sources, homogeneity of the light sources is important as well as that the fluorescent units inside the light source maintain their photophysical properties, which is compromised by dye aggregation. Here we propose DNA origami as a rigid scaffold to arrange dye molecules in a dense pixel array with high control of stoichiometry and dye-dye interactions. In order to find the highest labeling density in a DNA origami structure without influencing dye photophysics, we alter the distance of two ATTO647N dyes in single base pair steps and probe the dye-dye interactions on the single-molecule level. For small distances strong quenching in terms of intensity and fluorescence lifetime is observed. With increasing distance, we observe reduced quenching and molecular dynamics. However, energy transfer processes in the weak coupling regime still have a significant impact and can lead to quenching by singlet-dark-state-annihilation. Our study fills a gap of studying the interactions of dyes relevant for superresolution microscopy with dense labeling and for single-molecule biophysics. Incorporating these findings in a 3D DNA origami object will pave the way to bright and homogeneous DNA origami nanobeads.

DNA nanotechnology has developed into a state where the design and assembly of complex nanoscale structures has become fast, reliable, cost-effective, and accessible to non-experts. Nanometer-precise positioning of organic (dyes, biomolecules, etc.) and inorganic (metal nanoparticles, colloidal quantum dots, etc.) components on DNA nanostructures is straightforward and modular. In this perspective article, we identify the opportunities and challenges that DNA-assembled devices and materials are facing for optical antennas, metamaterials, and sensing applications. With the abilities of arranging hybrid materials in defined geometries, plasmonic effects will, for example, amplify molecular recognition transduction so that single-molecule events will be measureable with simple devices. On the larger scale, DNA nanotechnology has the potential of breaking the symmetry of common self-assembled functional materials creating pre-defined optical properties such as refractive index tuning, Bragg reflection and topological insulation.

In recent years, DNA nanotechnology has matured to enable robust production of complex nanostructures and hybrid materials. We have combined DNA nanotechnology with sensitive optical detection to create functional single-molecule devices that enable new applications in single-molecule biosensing and superresolution microscopy. Starting with superresolution nanorulers and brightness reference samples we determined the resolving power of superresolution microscopes and evaluated the sensitivity of smartphone cameras. To improve the sensitivity we created DNA origami optical antennas for metal enhanced fluorescence. The unique ability of our DNA origami nanoantennas to place molecular assays specifically in the plasmonic hotspot is used for detecting Zika-virus nucleic acids. The fluorescence enhancement is further exploited to achieve ultra-sensitive detection on low-cost devices such as a modified smartphone leading to the democratization of single-molecule detection.

There is a continuous demand for imaging probes offering excellent performance in various microscopy techniques for comprehensive investigations. of cellular processes by more than, one technique. FluoreScent nanodianiond gold nanoparticles (END Au) constitute a new class of "all in -one" hybrid particles providing.unique features fOr multimodal imaging including optical imaging, eleCtron microscopy, and, and potentially even quantum, sensing. COnfocal and optical coherence microscopy of the END Au allow fast investigations inside living cells via emission, scattering, and photothermal imaging techniques because, the END 'emission is not quenched by AuNPs. In electron microscopy, transmission electron microscopy (TEM) and. scanning transmission electron microscopy (STEM) analysis of END Au reveals greatly enhanced contrast due to the gold particles as well as an extraordinary flickering behavior in three-diniensional Cellular environments originating from the nanodiamonds. The unique multimodal imaging characteristics of END Au enable detailed studies inside cells ranging from statistical distributions at the entire cellular level (micrometers) down to the tracking of individual particles in sub cellular organelles (nanometers). Herein, the processes of endosomal membrane uptake and release of FNDs were elucidated for the first time by the imaging of individual END Au hybrid nanoparticles with single-particle resolution. Their convenient preparation, the availability of various surface groups, their flexible detection modalities, and their single-particle contrast in combination with the capability for endosomal penetration and low cytotoxicity make FND Au unique candidates for multimodal optical electronic imaging applications with great potential for emerging techniques, such as quantum sensing inside living cells.

For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution.

The application of commonly used force spectroscopy in biological systems is often limited by the need for an invasive tether connecting the molecules of interest to a bead or cantilever tip. Here we present a DNA origami‐based prototype in a comparative binding assay. It has the advantage of in situ readout without any physical connection to the macroscopic world. The seesaw‐like structure has a lever that is able to move freely relative to its base. Binding partners on each side force the structure into discrete and distinguishable conformations. Model experiments with competing DNA hybridisation reactions yielded a drastic shift towards the conformation with the stronger binding interaction. With reference DNA duplexes of tuneable length on one side, this device can be used to measure ligand interactions in comparative assays.

The DNA origami technique itself is considered a milestone of DNA nanotechnology and DNA origami nanorulers represent the first widespread application of this technique. DNA origami nanorulers are used to demonstrate the capabilities of techniques and are valuable training samples. They have meanwhile been developed for a multitude of microscopy methods including optical microscopy, atomic force microscopy, and electron microscopy, and their unique properties are further exploited to develop point-light sources, brightness references, nanophotonic test structures, and alignment tools for correlative microscopy. In this perspective, we provide an overview of the basics of DNA origami nanorulers and their increasing applications in fields of optical and especially super-resolution fluorescence microscopy. In addition, emerging applications of reference structures based on DNA origami are discussed together with recent developments.

Fluorescent dyes used for single-molecule spectroscopy can undergo millions of excitation-emission cycles before photobleaching. Due to the upconcentration of light in a plasmonic hotspot, the conditions for fluorescent dyes are even more demanding in DNA origami nanoantennas. Here, we briefly review the current state of fluorophore stabilization for single-molecule imaging and reveal additional factors relevant in the context of plasmonic fluorescence enhancement. We show that despite the improved photostability of single-molecule fluorophores by DNA origami nanoantennas, their performance in the intense electric fields in plasmonic hotspots is still limited by the underlying photophysical processes, such as formation of dim states and photoisomerization. These photophysical processes limit the photon count rates, increase heterogeneity and aggravate quantification of fluorescence enhancement factors. These factors also reduce the time resolution that can be achieved in biophysical single-molecule experiments. Finally, we show how the photophysics of a DNA hairpin assay with a fluorophore-quencher pair can be influenced by plasmonic DNA origami nanoantennas leading to implications for their use in fluorescence-based diagnostic assays. Especially, we show that such assays can produce false positive results by premature photobleaching of the dark quencher.

Fluorescence resonance energy transfer (FRET) has been instrumental in determining the structure and dynamics of biomolecules but distances above 8 nanometers are not accessible. However, with the advent and rapid development of super-resolution (SR) microscopy, distances between two fluorescent dyes below 20 nanometers can be resolved, which hitherto has been inaccessible for fluorescence microscopy approaches due to the limited resolving power of an optical imaging system that is determined by the fundamental laws of light diffraction (referred to as the diffraction limit). Therefore, the question arises whether SR microscopy can ultimately close the resolution gap between FRET and the diffraction limit and whether SR microscopy can be employed for the structural interrogation of proteins in the sub-20 nm range? Here, we show that the combination of DNA nanotechnology and single-molecule biochemistry allows the first step towards the investigation of the structural organization of a protein via SR microscopy. Limiting factors and possible future directions for the full implementation of SR microscopy as a structural tool are discussed.

We explore the potential of DNA nanotechnology for developing novel optical voltage sensing nanodevices that convert a local change of electric potential into optical signals. As a proof-of-concept of the sensing mechanism, we assembled voltage responsive DNA origami structures labeled with a single pair of FRET dyes. The DNA structures were reversibly immobilized on a nanocapillary tip and underwent controlled structural changes upon application of an electric field. The applied field was monitored through a change in FRET efficiency. By exchanging the position of a single dye, we could tune the voltage sensitivity of our DNA origami structure, demonstrating the flexibility and versatility of our approach. The experimental studies were complemented by coarse-grained simulations that characterized voltage-dependent elastic deformation of the DNA nanostructures and the associated change in the distance between the FRET pair. Our work opens a novel pathway for determining the mechanical properties of DNA origami structures and highlights potential applications of dynamic DNA nanostructures as voltage sensors.

In this contribution, we fabricate hybrid constructs based on a natural light-harvesting complex, peridinin–chlorophyll a–protein, coupled to dimer optical antennas self-assembled with the help of the DNA origami technique. This approach enables controlled positioning of individual complexes at the hotspot of the optical antennas based on large, colloidal gold and silver nanoparticles. Our approach allows us to selectively excite the different pigments present in the harvesting complex, reaching a fluorescence enhancement of 500-fold. This work expands the range of self-assembled functional hybrid constructs for harvesting sunlight and can be further developed for other pigment–proteins and proteins.

Optical nanoantennas are known to focus freely propagating light and reversely to mediate the emission of a light source located at the nanoantenna hotspot. These effects were previously exploited for fluorescence enhancement and single-molecule detection at elevated concentrations. We present a new generation of self-assembled DNA origami based optical nanoantennas with improved robustness, reduced interparticle distance, and optimized quantum-yield improvement to achieve more than 5000-fold fluorescence enhancement and single-molecule detection at 25 μM background fluorophore concentration. Besides outperforming lithographic optical antennas, DNA origami nanoantennas are additionally capable of incorporating single emitters or biomolecular assays at the antenna hotspot.

Forces in biological systems are typically investigated at the single-molecule level with atomic force microscopy or optical and magnetic tweezers, but these techniques suffer from limited data throughput and their requirement for a physical connection to the macroscopic world. We introduce a self-assembled nanoscopic force clamp built from DNA that operates autonomously and allows massive parallelization. Single-stranded DNA sections of an origami structure acted as entropic springs and exerted controlled tension in the low piconewton range on a molecular system, whose conformational transitions were monitored by single-molecule Förster resonance energy transfer. We used the conformer switching of a Holliday junction as a benchmark and studied the TATA-binding protein-induced bending of a DNA duplex under tension. The observed suppression of bending above 10 piconewtons provides further evidence of mechanosensitivity in gene regulation.

Because of the limited signal-to-background ratio, molecular diagnostics requires molecular amplification of the target molecules or molecular signal amplification after target recognition. For direct molecular detection, we demonstrate a purely physical fluorescence enhancement process which can elevate the fluorescence signal of single fluorescent dyes by several orders of magnitude. To this end, DNA origami-based optical antennas with a height of around 125 nm are used, which utilize metallic nanoparticles to create a hotspot where fluorescence signals are enhanced by plasmonic effects. By equipping the hotspot with a molecular beacon-like structure, we combine the plasmonic signal enhancement with a specific signal generation, leading to an enhanced and therefore easy to detect signal only in the presence of the specific target nucleic acid. Exemplified with Zika virus detection, we show the applicability of this approach by detecting Zika-specific artificial DNA and RNA both in buffer and in heat-inactivated human blood serum. We show the sensitivity against small nucleotide variations of this approach, allowing the discrimination of closely related pathogens. Furthermore, we show how the modularity offered by DNA nanotechnology enables multiplexing by incorporating orthogonal fluorescent labels for the simultaneous detection of different sequences. The achieved signal enhancement will allow technically simplified signal detection, paving the way for single molecule-based point-of-care diagnosis.

Sensing nucleic acids typically involves the recognition of a specific sequence and reporting by, for example, a fluorogenic reaction yielding one activated dye molecule per detected nucleic acid. Here, we show that after binding to a DNA origami track a bound DNA target (a “DNA walker”) can release the fluorescence of many molecules by acting as the catalyst of an enzymatic nicking reaction. As the walking kinetics sensitively depends on the walker sequence, the resulting brightness distribution of DNA origamis is a sequence fingerprint with single-nucleotide sensitivity. Using Monte Carlo simulations, we rationalize that the random self-avoiding walk is mainly terminated when steps to nearest neighbors are exhausted. Finally, we demonstrate that the DNA walker is also active in a plasmonic hotspot for fluorescence enhancement, indicating the potential of combining different amplification mechanisms enabled by the modularity of DNA nanotechnology.

Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

Resolution of emerging superresolution microscopy is commonly characterized by the width of a point-spread-function or by the localization accuracy of single molecules. In contrast, resolution is defined as the ability to separate two objects. Recently, DNA origamis have been proven as valuable scaffold for self-assembled nanorulers in superresolution microscopy. Here, we use DNA origami nanorulers to overcome the discrepancy of localizing single objects and separating two objects by resolving two docking sites at distances of 18, 12, and 6 nm by using the superresolution technique DNA PAINT(point accumulation for imaging in nanoscale topography). For the smallest distances, we reveal the influence of localization noise on the yield of resolvable structures that we rationalize by Monte Carlo simulations.

DNA origami used as a platform to study the bleaching behavior of four commonly used fluorophores in cw-STED nanoscopy., Superresolution microscopy is currently revolutionizing optical imaging. A key factor for getting images of highest quality is – besides a well-performing imaging system – the labeling of the sample. We compared the fluorescent dyes Abberior Star 488, Alexa 488, Chromeo 488 and Oregon Green 488 for use in continuous wave (cw-)STED microscopy in aqueous buffer and in a durable polymer matrix. To optimize comparability, we designed DNA origami standards labeled with the fluorescent dyes including a bead-like DNA origami with dyes focused on one spot and a DNA origami with two marks at a designed distance of ∼100 nm. Our data show that all dyes are well suited for cw-STED microscopy but that the optimal dye depends on the embedding medium. The precise comparison enabled by DNA origami nanorulers indicates that these structures have matured from the proof-of-concept to easily applicable tools in fluorescence microscopy.

Over the last decade, single-molecule detection has been successfully utilized in the life sciences and materials science. Yet, single-molecule measurements only yield meaningful results when working in a suitable, narrow concentration range. On the one hand, diffraction limits the minimal size of the observation volume in optical single-molecule measurements and consequently a sample must be adequately diluted so that only one molecule resides within the observation volume. On the other hand, at ultra-low concentrations relevant for sensing, the detection volume has to be increased in order to detect molecules in a reasonable timespan. This in turn results in the loss of an optimal signal-to-noise ratio necessary for single-molecule detection. This review discusses the requirements for effective single-molecule fluorescence applications, reflects on the motivation for the extension of the dynamic concentration range of single-molecule measurements and reviews various approaches that have been introduced recently to solve these issues. For the high-concentration limit, we identify four promising strategies including molecular confinement, optical observation volume reduction, temporal separation of signals and well-conceived experimental designs that specifically circumvent the high concentration limit. The low concentration limit is addressed by increasing the measurement speed, parallelization, signal amplification and preconcentration. The further development of these ideas will expand our possibilities to interrogate research questions with the clarity and precision provided only by the single-molecule approach.

Obtaining quantitative information about molecular assemblies with high spatial and temporal resolution is a challenging task in fluorescence microscopy. Single-molecule techniques build on the ability to count molecules one by one. Here, a method is presented that extends recent approaches to analyze the statistics of coincidently emitted photons to enable reliable counting of molecules in the range of 1-20. This method does not require photochemistry such as blinking or bleaching. DNA origami structures are labeled with up to 36 dye molecules as a new evaluation tool to characterize this counting by a photon statistics approach. Labeled DNA origami has a well-defined labeling stoichiometry and ensures equal brightness for all dyes incorporated. Bias and precision of the estimating algorithm are determined, along with the minimal acquisition time required for robust estimation. Complexes containing up to 18 molecules can be investigated non-invasively within 150 ms. The method might become a quantifying add-on for confocal microscopes and could be especially powerful in combination with STED/RESOLFT-type microscopy.

While nanophotonic devices are unfolding their potential for single-molecule fluorescence studies, metallic quenching and steric hindrance, occurring within these structures, raise the desire for site-specific immobilization of the molecule of interest. Here, we refine the single-molecule cut-and-paste technique by optical superresolution routines to immobilize single fluorescent molecules in the center of nanoapertures. By comparing their fluorescence lifetime and intensity to stochastically immobilized fluorophores, we characterize the electrodynamic environment in these nanoapertures and proof the nanometer precision of our loading method.

In this work, a multifunctional template for selective formation of fluorescent silver nanoclusters (AgNCs) or copper nanoparticles (CuNPs) is put forward. This dumbbell-shaped (DS) DNA template is made up of two cytosine hairpin loops and an adenine-thymine-rich double-helical stem which is closed by the loops. The cytosine loops act as specific regions for the growth of AgNCs, and the double-helical stem serves as template for the CuNPs formation. By carefully investigating the sequence and length of DS DNA, we present the optimal design of the template. Benefiting from the smart design and facile synthesis, a simple, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection is proposed. Through the systematic comparison, it is found that the strategy based on CuNPs formation is more sensitive for ATP assay than that based on AgNCs synthesis, and the detection limitation was found to be 81 pM. What's more, the CuNPs formation-based method is successfully applied in the detection of ATP in human serum as well as the determination of cellular ATP. In addition to small target molecule, the sensing strategy was also extended to the detection of biomacromolecule (DNA), which illustrates the generality of this biosensor.

We study the distance-dependent quenching of fluorescence due to a metallic nanoparticle in proximity of a fluorophore. In our single-molecule measurements, we achieve excellent control over structure and stoichiometry by using self-assembled DNA structures (DNA origami) as a breadboard where both the fluorophore and the 10 nm metallic nanoparticle are positioned with nanometer precision. The single-molecule spectroscopy method employed here reports on the co-localization of particle and dye, while fluorescence lifetime imaging is used to directly obtain the correlation of intensity and fluorescence lifetime for varying particle to dye distances. Our data can be well explained by exact calculations that include dipole dipole orientation and distances. Fitting with a more practical model for nanosurface energy transfer yields 10.4 nm as the characteristic distance of 50% energy transfer. The use of DNA nanotechnology together with minimal sample usage by attaching the particles to the DNA origami directly on the microscope coverslip paves the way for more complex experiments exploiting dye nanoparticle interactions. Fil: Acuna, Guillermo P.. Technische Universität Braunschweig; Alemania Fil: Bucher, Martina. Ludwig Maximilians Universitat; Alemania Fil: Stein, Ingo H.. Ludwig Maximilians Universitat; Alemania Fil: Steinhauer, Christian. Ludwig Maximilians Universitat; Alemania Fil: Kuzyk, Anton. Technische Universitat Munchen; Alemania Fil: Holzmeister, Phil. Technische Universität Braunschweig; Alemania Fil: Schreiber, Robert. Ludwig Maximilians Universitat; Alemania Fil: Moroz, Alexander. Fil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Liedl, Tim. Ludwig Maximilians Universitat; Alemania Fil: Simmel, Friedrich C.. Technische Universitat Munchen; Alemania Fil: Tinnefeld, Philip. Technische Universität Braunschweig; Alemania

Fluorescence resonance energy transfer (FRET) has become a work-horse for distance measurements on the nanometer scale and between single molecules. Recent model systems for the FRET distance dependence such as polyprolines and dsDNA suffered from limited persistence lengths and sample heterogeneity. We designed a series of rigid DNA origami blocks where each block is labeled with one donor and one acceptor at distances ranging between 2.5 and 14 nm. Since all dyes are attached in one plane to the top surface of the origami block, static effects of linker lengths cancel out in contrast to commonly used dsDNA. We used single-molecule spectroscopy to compare the origami-based ruler to dsDNA and found that the origami blocks directly yield the expected distance dependence of energy transfer since the influence of the linkers on the donor―acceptor distance is significantly reduced. Based on a simple geometric model for the inter-dye distances on the origami block, the Forster radius R o could directly be determined from the distance dependence of energy transfer yielding R 0 = 5.3 ± 0.3 nm for the Cy3―Cy5 pair.

DNA origami is a powerful method for the programmable assembly of nanoscale molecular structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resolution becomes increasingly important. We present a single-molecule assay for the study of binding and unbinding kinetics on DNA origami. We find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, we exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topography) imaging with

In this paper we experimentally combine a recently developed AFM-based molecule-by-molecule assembly (single-molecule cut-and-paste, SMCP) with subdiffraction resolution fluorescence imaging. Using “Blink-Microscopy”, which exploits the fluctuating emission of single molecules for the reconstruction of superresolution images, we resolved SMCP assembled structures with features below the diffraction limit. Artificial line patterns then served as calibration structures to characterize parameters, such as the labeling density, that can influence resolution of Blink-Microscopy besides the localization precision of a single molecule. Finally, we experimentally utilized the adjustability of blink parameters to demonstrate the general connection of photophysical parameters with spatial resolution and acquisition time in superresolution microscopy.

In recent years fluorescence resonance energy transfer (FRET) has widely been used to measure distances, binding, and distance dynamics at the single-molecule (sm) level. Some basic constraints of smFRET are the limited distance resolution owing to low photon statistics and the restriction to high affinity interactions. We demonstrate that by confining molecules in nanopipets with an inner diameter of approximately 100 nm at the tip, FRET can be measured with improved photon statistics and at up to 50-fold higher concentrations. The flow of the donor/acceptor (Cy3B/ATTO647N) labeled double-stranded DNA conjugates was established by electrokinetic forces. Because of the small inner diameter of the nanopipet, every molecule passing the tip is detected applying alternating laser excitation (ALEX). Thus, the technique offers the advantage to study interactions with smaller association constants (10(9) M-1) using minute sample amounts (5 microL). The improved photon statistics reduces shot-noise contributions and results in sharper FRET distributions. Experimental results are supported by Monte Carlo simulations which also explain the occurrence of two populations in burst size distributions measured in nanopipet experiments. Because of the confinement of the molecules in nanopipets, the widths of FRET histograms are reduced to a degree where shot-noise is not the only limiting factor but also conformational dynamics of the linkers used to attach the chromophores have to be considered. In addition, our experiments emphasize the influence of photoinduced dark states on both the mean energy transfer efficiency and the width of FRET histograms.

We used electron-beam lithography to fabricate chemical nanostructures, i.e, amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of similar to2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of similar to1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to similar to2. The S/B-ratio of the fluorescence signals could be further increased to similar to4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes. (C) 2004 Elsevier B.V. All rights reserved.

Fluorescence resonance energy transfer (FRET) represents a mechanism to transport light energy at the nanoscale, as exemplified by nature's light-harvesting complexes. Here we used DNA origami to arrange fluorophores that transport excited-state energy from an input dye to an output dye. We demonstrate that energy-transfer paths can be controlled on the single-molecule level by the presence of a "jumper" dye that directs the excited-state energy either to a red or to an IR output dye. We used single-molecule four-color FRET with alternating laser excitation to sort subpopulations and to visualize the control of energy transfer.

Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d.

Nanotechnology is challenged by the need to connect top-down produced nanostructures with the bottom-up world of chemistry. A nanobiotechnological prime example is the positioning of single polymerase molecules in small holes in metal films, so-called zeromode waveguides (ZMWs), which is required for single-molecule real-time DNA sequencing. In this work, we present nanoadapters made of DNA (DNA origami) that match the size of the holes so that exactly one nanoadapter fits in each hole. By site-selective functionalization of the DNA origami nanoadapters, we placed single dye molecules in the ZMWs, thus optimizing the hole usage and improving the photophysical properties of dyes compared to stochastically immobilized molecules.

The amount of information obtainable from a fluorescence-based measurement is limited by photobleaching: Irreversible photochemical reactions either render the molecules nonfluorescent or shift their absorption and/or emission spectra outside the working range. Photobleaching is evidenced as a decrease of fluorescence intensity with time, or in the case of single molecule measurements, as an abrupt, single-step interruption of the fluorescence emission that determines the end of the experiment. Reducing photobleaching is central for improving fluorescence (functional) imaging, single molecule tracking, and fluorescence-based biosensors and assays. In this single molecule study, we use DNA self-assembly to produce hybrid nanostructures containing individual fluorophores and gold nanoparticles at a controlled separation distance of 8.5 nm. By changing the nanoparticles? size we are able to systematically increase the mean number of photons emitted by the fluorophores before photobleaching. Fil: Pellegrotti, Jesica Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina Fil: Acuña, Guillermo. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania Fil: Puchkova, Anastasiya. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania Fil: Holzmeister, Phil. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania Fil: Gietl, Andreas. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania Fil: Lalkens, Birka. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania Fil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina Fil: Tinnefeld, Philip. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemania

The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution.

Summary For detailed experimental studies about the electromagnetic coupling between plasmonic nanoparticles, precisely-controlled nanoparticle groupings with adjustable inter-particle distance would be required. In the present review article, the possibilities of generating such groupings by using biological and polymeric spacers in between the nanoparticles are described. In particular, streptavidin–biotin and DNA are discussed as biological spacers, and layer-by-layer (LbL) assembled films as polymeric spacers. The concepts are visualized by experimental data, which also point out the pitfalls and current limits of such approaches. Calculation results reveal that while small variations in the diameter of plasmonic cores lead to small changes, small variations in the inter-particle distance due to soft linkers result in significant changes in plasmonic coupling. In the case of protein-based spacers, nonspecific adsorption has to be considered as an obstacle for the formation of controlled groupings.